I. Field of the Invention
This invention relates to techniques used for the polymerization and amplification of sequences of nucleic acids of prokaryotic and eukaryotic origin involving the polymerase chain reaction or the ligase chain reaction. More particularly, the invention relates to a process and apparatus for carrying out such techniques.
II. Background Art
The amplification of nucleic acid sequences by means of a chain reaction technique using a nucleic acid polymerase enzyme has been developed over the last several years to enable minute amounts of nucleic acids, e.g. DNA, to be copied in quantity to an extent suitable for detection and analysis. The procedure is described, for example, in U.S. Pat. Nos. 4,683,195, 4,683,202, 5,436,149, 5,405,774, 5,340,728, 5,338,671 and 4,965,188, the disclosures of which are incorporated herein by reference. Recently, a related chain reaction technique involving a nucleic acid ligase enzyme has also been developed.
The polymerase chain reaction (PCR) procedure is briefly explained as follows. In nature, the replication of duplex DNA is carried out by the key enzyme DNA polymerase that has two activities, namely:
a) 5'-3' polymerase activity, i.e. the addition of new nucleotides to the growing strand at the 3-prime end of a primer, probe (labeled primer) or synthesizing strand; and PA1 b) 3'-5' exonuclease activity, i.e. the removal of nucleotides from the 3-prime end which may be misincorporated into the synthesizing strand. PA1 a) A single stranded DNA or RNA template. PA1 b) An oligonucleotide (primer or probe) having a nucleotide at the 3'-prime end carrying a hydroxy group at the 3rd position of the sugar molecule and carrying a base molecule which is complementary to the corresponding base molecule on the single stranded template strand. PA1 c) A set of synthetic nucleotides (dTTP, dCTP, dGTP, dATP) activated by magnesium ions at pH 8.3-pH 8.4. PA1 d) A DNA polymerase enzyme which has the ability to add new nucleotides at the 3-prime end of the primer (or to the growing chain) via phosphodiester bonds. PA1 a) Denaturing--Denaturing is a process whereby the individual strands of the DNA are separated by breaking the hydrogen bonds across the bases of the complementary nucleotides. At present, this is normally achieved by heating the DNA to near the boiling point of water (more specifically, to a "melting" temperature greater than 94.degree. C.). PA1 b) Annealing--This is a process whereby synthetic oligonucleotide primers or probes (each normally containing about 20 nucleotides) bind to complementary sequences of any single DNA strand present by the formation of hydrogen bonds across the bases of the complementary nucleotides. Pairs of primers are generally used, one for each strand of the DNA, flanking a sequence of interest, normally about 100 to 5,000 base pairs (bp) in length. The temperature at which annealing takes place is normally 37.degree. to 70.degree. C. PA1 c) Polymerization--This involves the addition of new nucleotides at the 3'-prime end of the primer by the formation of phosphodiester bonds in the presence of Taq DNA polymerase. The polymerization reaction normally takes place at a temperature of about 72.degree. C. PA1 DNA polymerase 1 E. coli lambda lysogen NM 864; PA1 DNA polymerase 1 Large Klenow fragment which lacks 5'-3' exonuclease activity; PA1 AMV reverse transcriptase; PA1 Murine reverse transcriptase; PA1 M-MLV (Moloney murine Leukemia virus) reverse transcriptase expressed in E. coli as pol gene; PA1 SP6 RNA polymerase; PA1 T3 RNA polymerase; PA1 T7 RNA polymerase; PA1 T4 DNA polymerase; and PA1 T7 DNA polymerase. PA1 E. coli DNA ligase, PA1 T4 DNA ligase, and PA1 T4 RNA ligase. PA1 recombinant pfu DNA ligase from Pyrococcus furiosus. PA1 Bacteria: Bacillus subtilis PA1 Blue green algae: Plectonema nostocorum Arthrospira plantenesis PA1 Fungi: Penicillium variables Fusarium bullatum Fusarium oxysporium. PA1 a) Identification of microbial pathogens. PA1 b) Identification of mutations and hereditary diseases. PA1 c) Quantification of gene expression in cells. PA1 d) DNA sequencing. PA1 e) RNA sequencing. PA1 a) There is no need for an expensive thermocycler (the apparatus conventionally used for the PCR/LCR procedure) and therefore the required capital cost is less. This may make the equipment sufficiently cost effective that the process may carried out in a decentralized testing facility (such as doctors' offices). PA1 b) The apparatus may be designed to hold one or more removable housings (referred to herein as cassettes) each enclosing multiple containers for the various steps of the PCR/LCR method. When multiple cassettes are used, it is possible to carry out two or more PCR/LCR reactions on different substrates simultaneously, e.g. to analyze more than one type of sample at any one time on the same equipment using different cassettes, e.g. E. coli and Salmonella. Such cassettes may be produced as disposable units that may be kept sealed and provided with sealable inlets/outlets. PA1 c) The number of samples analyzed at any one time can be varied. The equipment may have capacity to deal with:
The enzyme can be used for artificial replication of DNA by employing four basic elements:
Given the fact that DNA occurs naturally as two complementary strands joined by hydrogen bonds, and that both strands of DNA may function as templates for the synthesis of new strands by polymerization, it is possible to duplicate a specific segment of DNA by using a pair of complementary oligonucleotides as primers which can bind across (i.e. on opposite sides of) the segment of interest (the so-called target sequence). It is known that, to facilitate the binding of primers to the template, the hydrogen bonds across the double stranded DNA first have to be broken and the single strands have to be separated. Conventionally, this is carried out by heating the template to a temperature greater than 94.degree. C. and, when the sample cools, allowing the oligonucleotide primers to bind to (anneal with) complementary regions on the template. This is followed by the polymerase reaction which causes new double stranded DNA to be formed by nucleotide polymerization using the target sequence as a template for the selection of nucleotides for the new complementary sequence. The polymerase chain reaction extends this concept by making more copies of the target sequence by repeating the sequence of steps by cycling the temperature around the denaturing point while maintaining the reactants in a single chamber or reaction zone. Since normal DNA polymerase is heat sensitive (i.e. thermolabile), and is deactivated if heated to the temperature required for the separation of strands of DNA, it was initially necessary to add an influx of fresh polymerase prior to the synthesis step for each cycle. To overcome this limitation, a heat stable polymerase was isolated from Thermophilus acquaricus. Recently, the gene for thermostable polymerase has been cloned in expression vectors, and a recombinant heat stable enzyme produced and made commercially available.
The most common heat stable enzyme of this type is referred to as Taq DNA polymerase from Thermophilus acquaticus, but others are also known, e.g. Tth DNA polymerase from Thermophilus thermophiles and Tth DNA polymerase from Thermophilus flavus. The existence of such enzymes makes it possible to combine the starting materials and reactants in a single reaction zone or chamber and to cycle the temperature above and below 94.degree. C. to produce the steps required for PCR without further additions of polymerase.
Thus, at present, the polymerase chain reaction in its most common form has three stages. They are:
When this cycle is repeated many times (normally at least 30 times with each cycle typically lasting from 3 to 5 minutes), a detectable amount of the target DNA is produced.
The ligase chain reaction (LCR) is similar to PCR, except that short stretches of nucleic acid (probes), bound to a target sequence template, are joined together by a nucleic acid ligase enzyme. LCR is often used to distinguish between normal DNA of known sequence and mutant DNA. A pair of DNA probes having a sequence which, taken together, are complementary to the expected target nucleic acid are produced and brought into contact with the target sequence in the presence of a nucleic acid ligase enzyme. If double stranded target DNA is denatured and allowed to anneal with the probes, the probes will bind to the target DNA with ends adjacent to each other. The ligase enzyme then binds the probe DNA to form a single strand comprising both probes. If the target DNA differs from the expected sequence, the probes will not bind properly and the ligase enzyme will not be able to form the combined single strand. Repeated cycles amplify the combined single strand (if formed) which can be distinguished from the probes themselves by nucleotide length and by the presence of markers from both probes. If one of the probes is bound to a solid support and the other is not, the combined single strand will also be bound to the solid support and, after washing to remove the unbound probe, the presence of the combined strand can be detected by the presence of the marker used for the unbound probe. As in the case of PCR, LCR is carried out by repeated thermal cycles to cause denaturing and annealing of the DNA, so a thermostable ligase enzyme is required.
In spite of the feasibility of PCR and LCR, the widespread use of these techniques has been limited somewhat by the high capital cost of the required thermocycler apparatus, which tends to be complex in design and construction, and the high cost of the available thermostable polymerases or ligases. Therefore, current PCR technology often necessitates centralized testing at sites which are often distant from the point of sampling. This arrangement not only increases the cost but also delays the reporting of results. It would therefore be desirable to provide a PCR method and apparatus that could be used less expensively and made more widely available.